Article available online at http://www.idealibrary.com on Microbial Pathogenesis 2000; 28: 363–372

PATHOGENESISMICROBIAL

doi:10.1006/mpat.2000.0356

Characterization of surface layer proteinsfrom different Clostridium difficile clinicalisolatesMarina Cerquettia, Agnese Molinarib, Annalisa Sebastianellia,Marco Diociaiutib, Raffaele Petruzzellic, Concetta Capod &Paola Mastrantonioa∗aDepartment of Bacteriology and Medical Mycology and bDepartment of Ultrastructures, IstitutoSuperiore di Sanita, viale Regina Elena 299, 00161 Rome, Italy, cDepartment of Biomedical Science,University of G. D’Annunzio, 66100 Chieti, Italy and dDepartment of Biology, University of Rome TorVergata, via Della Ricerca Scientifica, 00133, Rome, Italy

(Received October 8, 1999; accepted in revised form February 16, 2000)

In a previous study we suggested that two surface proteins of a Clostridium difficile strain wereinvolved in the formation of a regularly assembled surface layer (S-layer) external to the cell wall.In the present paper six C. difficile strains isolated from cases and healthy carriers were studied.By using freeze-etching and negative staining techniques two superimposed structurally differentlattices were detected on the cell surface of the different C. difficile strains. In each strain, theouter S-layer lattice was arranged in a square symmetry and the inner S-layer lattice in hexagonalsymmetry. The S-layer proteins from the different strains were isolated and characterized. Eachstrain showed two distinct S-layer glycoproteins ranging in molecular mass 36–56 kDa. Antigeniccross-reactivity among the S-layer proteins of higher molecular masses extracted from each strainwas demonstrated whereas no antigenic relationship was observed among the different S-layerproteins of lower molecular masses. N-terminal sequence analysis showed the presence of commonstructural motifs conserved among the high S-layer proteins as well as among the low S-layerproteins. These data indicate that the presence of S-layer on C. difficile strains is common andthat its glycoprotein subunits show a certain degree of heterogeneity. 2000 Academic Press

Key words: Clostridium difficile proteins; surface layer (S-layer).

diarrhoea (AAD), has been related to the pro-Introductionduction of the toxins A and B [1, 2]. Nevertheless,other virulence factors, mainly those which can

The pathogenicity of Clostridium difficile, the eti- promote the bacterial colonization of the host,ological agent of pseudomembranous colitis may play a fundamental role. Previous studies(PMC) and most cases of antibiotic associated [3, 4] demonstrated the immunodominant char-

acter of some surface proteins of C. difficile inpatients with AAD and their common features∗Author for correspondence: E-mail: pmastran@iss.it

0882–4010/00/060363+10 $35.00/0 2000 Academic Press

364 M. Cerquetti et al.

with the surface proteins involved in the forma- or oblique) symmetry model. To discriminatebetween the oblique symmetry and the squaretion of the crystalline surface layer (S-layer) in

other bacteria. one, the b/a value (ratio of the length of thebase vectors a→ and b→) must be considered.S-layers have been observed as the outermost

cell envelope in a variety of Gram-positive and In the oblique model the distance between per-pendicular points must be significantly different,Gram-negative bacteria [5–7]. They are com-

posed of identical protein or glycoprotein sub- that is b/a about 1.6 while in the square modelb/a must be about 1 [13]. The results of theunits forming oblique, square or hexagonal

lattices on the bacterial cell surface. Various freeze-etching observations showing b/a valuesaround 1 (Fig. 1 legend), clearly indicate thefunctions have been proposed for S-layers in-

cluding shape maintenance, molecular sieving presence of square ordered lattices with similarbut not the same centre to centre spacing on theor phage fixation. In pathogenic bacteria the S-

layer represents an important interface between cell surface of the six different C. difficile strains.the bacterial cell and its host. In some cases S-layers were identified as virulence factors pro-tecting pathogenic bacteria against complement Electron microscopy of isolated cell-wall

fragments of C. difficile strains by negativekilling, facilitating binding of the bacterium tohost molecules or enhancing its ability to as- stainingsociate with macrophages [7–9].

The presence of a regular arranged S-layer The isolated cell-wall fragments of the six C.difficile strains examined by electron microscopyin two C. difficile strains has been previously

described [10, 11]. Our preliminary observations after negative staining showed the presence ofa hexagonally ordered arrangement of mor-also indicated the occurrence of a crystalline S-

layer on the cell surface of a C. difficile strain phological units with lattice spacing parametersranging 8.3–10 nm (data not shown). Fig. 2[12]. These findings were considered stimulating

enough to extend investigations on toxigenic shows negative staining images of a cell-wallfragment of C. difficile C253 prototype strain (a)and non toxigenic C. difficile isolates from epi-

demiologically unrelated patients with AAD and together with the relative Fourier pattern (b),revealing an hexagonal symmetry (angle valuehealthy carriers. In the present study six C.

difficile strains were analysed by freeze-etching 60°) with lattice spacing of about 10 nm. TheFourier pattern appeared to be more complexand negative staining techniques to investigate

the presence and to characterize the structure of than a basic hexagonal arrangement due to thepresence of multiple spots.an S-layer on their surfaces. With the aim to

reveal relationship among S-layer proteins ex-tracted from different C. difficile strains, the bio-chemical and immunological characterization of In vitro self-assembly of C. difficile C253

S-layer proteinsthe isolated S-layer proteins were also per-formed.

The treatment of cell-wall fragments of C. difficileC253 with 8 M urea resulted in the completeremoval of the hexagonal ordered lattice asjudged by negative staining (data not shown).ResultsSDS-PAGE of the urea extract showed the pres-ence of two proteins with molecular masses ofElectron microscopy of C. difficile whole

cells by freeze-etching 47 and 36 kDa, respectively.When the urea extract containing both pro-

teins was dialysed to remove the dissociatingThe presence of a crystalline regular array wasvisualized by freeze-etching on the cell surface agent, sheet-like fragments showing a regular

lattice pattern were recovered. As shown in Fig.of all six C. difficile strains examined (Fig. 1).To identify the symmetries of the six lattices 2(c) and (d) a hexagonal symmetry (angle value

60°) with lattice spacing of about 10 nm wasobserved, FFT analysis was performed. The mostfrequent angle values measured in the reciprocal revealed by electron microscopy after negative

staining. No regular arrayed structures werespaces relative to the different C. difficile strainswere 45°, 90° and 135° (data not shown). These observed using the two proteins purified by

HPLC: both the two mixed subunits and eachangles are characteristic of the tetragonal (square

Clostridium difficile surface layer proteins 365

Figure 1. S-layer lattices of different C. difficile strains. Freeze-etched preparations of whole bacterial cellsdisplayed the square lattice molecular arrangement. The base-vector intensity (a) and (b) for each latticewas: C253 a: 10.5±1.6, b: 13.0±0.8; AN18 a: 11.3±2.3, b: 13.0±1.2; PA79 a: 11.8±1.4, b: 13.1±1.1; PX1394a: 10.4±1.2, b: 13.0±0.8; P5 a: 12.0±1.1, b: 12.9±0.7; 1699 a: 11.6±2.3, b: 12.2±2.9. Each value (nm) wasthe mean and standard deviation of 30 measurements. Bar=56 nm.

366 M. Cerquetti et al.

(a) (b)

(c) (d)

Figure 2. Negative staining preparations of cell-wall fragments (a) and of reassembled urea extracted S-layerproteins (c) from C. difficile C253. Negative staining preparations of the native cell-wall and of the reassembledS-layer proteins revealed a hexagonal arrangement of the subunits. The Fourier transform patterns are shownin (b) and (d). Bar=35 nm.

single subunit alone failed to reassemble any masses ranging 36–56 kDa [Fig. 3(a)]. Theproteins of higher molecular masses extractedregular array (data not shown).from different strains will now be referred to ashigh S-layer proteins, while the proteins of lowermolecular masses as low S-layer proteins.

When the same urea extracts were tested byComparison of the S-layer proteins among the DIG glycan/protein double labelling kit, twoC. difficile strains distinct glycoprotein bands for each strain could

be visualized indicating that all of the S-layerTo simplify the procedure, the extraction of the proteins are glycosylated [Fig. 3(b)].S-layer protein subunits from each C. difficile Western immunoblot analysis, using rabbitstrain was achieved by direct treatment of the antisera against the 36 and 47 kDa S-layer pro-whole cells with 8 M urea. Each of the six urea teins of C. difficile C253, was performed to in-extracts, examined by SDS-PAGE, showed the vestigate the antigenic relationship among the

S-layer proteins isolated from the six C. difficilepresence of two distinct proteins with molecular

Clostridium difficile surface layer proteins 367

97.4 –

7

kDa

1

66.2 –

45.0 –

31.0 –

21.5 –

14.4 –

(a)

65432

97.4 –

7

kDa

1

66.2 –

45.0 –

31.0 –

21.5 –

14.4 –

(b)

65432 8

Figure 3. SDS-PAGE (12% acrylamide gel) (a) and analysis by the DIG glycan/protein double labelling kit(b) of urea extract from different C. difficile strains. (a) lanes: 1, low-molecular weight standards; 2, C253; 3,AN18; 4, PA79; 5, PX1394; 6, P5; 7, 1699. Approximately 15 lg of protein for each urea extract was loaded.Each strain showed two distinct S-layer proteins migrating approximately at: 36 and 47 kDa (C253, AN18),40 and 52 kDa (PA79), 35 and 54 kDa (PX1394), 39 and 56 kDa (P5), 43 and 46 kDa (1699). (b) lanes: 1, controlglycoprotein fetuin (0.2 lg); 2, control protein creatinase (3 lg); 3, C253; 4, AN18; 5, PA79; 6, PX1394; 7, P5;8, 1699; the leftmost line shows the molecular weight standards. Approximately 3 lg of protein for eachurea extract was loaded. In the urea extract of each strain two distinct glycoprotein bands are visible.

112 –

7

kDa

1

84 –

53 –

34 –

28 –

20 –

(a)

65432

112 –

7

kDa

1

84 –

53 –

34 –

28 –

20 –

(b)

65432

Figure 4. Western immunoblot analysis of urea extracted S-layer proteins from different C. difficile strainsprobed with rabbit antisera to the 47 kDa S-layer protein (a) and to the 36 kDa S-layer protein (b). Lanes: 1,Prestained low-molecular weight standards; 2, C253; 3, AN18; 4, PA79; 5, PX1394; 6, P5, 7, 1699. Approximately5 lg of protein for each sample was loaded. Sera were diluted 1:1000. The antiserum to the 47 kDa proteinreacted with all the high S-layer proteins. In contrast, the antiserum against the 36 kDa protein reacted onlywith the 36 kDa protein bands of strains C253 and AN18.

strains. As shown in Fig. 4 the antiserum to the N-terminal sequence analysis of the S-layer proteins from C. difficile strains47 kDa exhibited strong antigenic reactivity with

all the high S-layer proteins. Conversely, theantiserum to the 36 kDa protein did not re- The sequences of about 30 N-terminal amino

acids of the S-layer proteins from C. difficile C253,cognize any S-layer protein with different mo-lecular mass but reacted only with the 36 kDa PA79, PX1394, P5, 1699 are shown in Fig. 5.

The comparison of the amino acid sequencesprotein extracted from the strains C253 andAN18. documented some degree of identity among the

368 M. Cerquetti et al.

Figure 5. N-terminal amino acid sequences of S-layer proteins from different C. difficile strains. Commonstructural motifs are conserved among the low S-layer proteins (on the left) and among the high S-layerproteins (on the right) isolated from the different C. difficile strains. The common motifs are underlined.

high as well as among the low S-layer proteins. A revealed by freeze-etching of C. difficile wholecells, a hexagonally ordered lattice becameshort sequence of four amino acids TyrThrValValvisible by negative staining on the isolatedwas found to be conserved among the low S-cell-wall fragments. This unexpected result islayer proteins. Similarly, two common structuralsimilar to data of Sara et al. [19] whichmotifs constituted by LysXxxValXxxLys anddescribed the occurrence of two superimposedLysXxxLysAsp (Xxx represents a hydrophobicstructurally different S-layers in two strains ofresidue) characterized the high S-layer proteins.Bacillus brevis. They also observed differentNo identity was found between the two S-layersymmetries by freeze-etching of whole cellsproteins extracted from the same strain. A searchand by negative staining of the cell-wall frag-for sequence data bases to find similarity withments and they concluded that in the negativeS-layer proteins of other bacteria did not revealstained preparations the inner thicker S-layerany conservative character.apparently masked the much thinner outer S-layer which was revealed by freeze-etchingtechnique, more sensitive to the surface to-pology. Accordingly, our data suggest that theDiscussion and ConclusionsS-layers of the C. difficile strains examined areconstituted by two superimposed S-layers, each

In this study, four toxigenic and two non-toxi- composed by a different protein: the outergenic strains belonging to different elec- arranged in tetragonal symmetry and the innertrophoretic types and isolated from cases and in hexagonal symmetry.healthy carriers were studied to investigate the In vitro self-assembly experiments dem-presence of an S-layer on their outermost sur- onstrate that the two proteins extracted byfaces. Image analysis performed on the freeze- urea from C. difficile C253 are in fact theetched samples clearly revealed the presence of components of the S-layer. The self-assembleda squarely arranged S-layer in each of the six layer possessed a hexagonally arrayed patternC. difficile strains examined, including the non- with lattice parameter of about 10 nm, whichtoxigenic strains isolated from an adult healthy was characteristic for the native S-layer ob-carrier and a child without diarrhoea, re- served on the cell-wall fragments of the strainspectively. after negative staining. It appeared to be

Treatment of C. difficile strains with 8 M urea a simplified version of the native cell-wallled to the extraction of two distinct S-layer pro- arrangement due perhaps to the lack in theteins from each strain. These proteins differed former of the underlying cell-wall. Moreover,in molecular mass from strain to strain except the native ordered structure on cell-walls dis-those from two isolates not epidemiologically appeared after urea treatment. On the contrary,related but belonging to the same electrophoretic purified 36 and 47 kDa proteins, obtained bytype, characteristic of strains from nosocomial reversed phase HPLC, failed to reassemble inoutbreaks [14]. A size heterogeneity of the S- vitro either separately or in mixture. Thelayer proteins even among strains belonging purification procedures probably caused theto the same bacterial species has already been loss of the self-assembling ability of the proteindescribed [15–17]. Even if most S-layers are com- subunits as reported also by other authorsposed of a single protein or glycoprotein species, [20].the presence in some C. difficile strains of an S- All the S-layer proteins extracted from C. di-layer composed of two distinct proteins has fficile strains appeared to be glycosylated. Thisalready been reported by other authors [11, 18]. result confirms our preliminary studies on pep-

tide glycosylation of digest solutions of the 47In this study, contrary to the square lattice

Clostridium difficile surface layer proteins 369

and 36 kDa S-layer proteins of C. difficile C253 Materials and Methods[21].

Western immunoblot analysis with the rabbit Bacterial strains and growth conditionsantisera against the 47 and 36 kDa S-layer pro-teins of C. difficile C253 showed the antigenic Six C. difficile strains isolated from variousrelatedness among the S-layer proteins isolated sources belonging to different electrophoreticfrom the six C. difficile strains. The results in- types [14, 24] were used in this study. Threedicate the presence of two distinct families of C. toxigenic strains were isolated from cases ofdifficile S-layer proteins: the first constituted by AAD not epidemiologically related (C253,the antigenically related high S-layer proteins AN18, PX1394, this last kindly provided by I. R.and the second composed of the non cross- Poxton, University of Edinburgh Medicalreactive low S-layer proteins. Therefore, each C. School, Edinburgh), one toxigenic strain from adifficile strain carries on its surface one high S- diarrhoeic neonate (PA79) and two non-toxi-layer protein with antigenic similarities with the genic strains from a healthy adult (1699) and acorresponding high S-layer proteins of other hospitalized child without intestinal symptomsC. difficile strains and one low S-layer protein (P5). Bacteria from an overnight culture wereantigenically unique in each strain. Antigenic grown anaerobically on proteose peptone yeastsimilarities or heterogeneity among S-layer pro- extract (PPY) broth (Oxoid S.p.A., Milan, Italy)teins within the same bacterial species has al- for 18 h at 37°C, as previously described [14].ready been reported [15–17, 22, 23].

The existence of two separate families of C.difficile S-layer proteins was confirmed by data Preparation of cell-wallsfrom N-terminal sequence analysis. Each family,constituted by the high or the low S-layer pro-

Bacterial cells were harvested by centrifugationteins, shared at least one common structural

at 5800×g for 30 min and washed once withmotif, which has not been previously recognized

50 mM Tris–HCl pH 7.4 at 4°C. The washed cellin the primary structure of other bacterial S-

pellet was suspended in cold buffer and thenlayer proteins. Different from other bacterial

broken by ultrasonication (three 1 min burstsspecies, in which the aminoterminal regions of

with 30 s cooling periods in between). Unbrokenthe S-layer proteins from different strains are

cells were removed by low speed centrifugationhighly conserved [15, 17], only little identity

and the cell-wall fragments were recovered byseems to exist among C. difficile S-layer proteins.

centrifugation at 13 000×g for 15 min. For elec-A search in the sequence data base failed to

tron microscopy observation, the crude cell en-reveal any significant homology between C. dif-

velopes were immediately fixed with 2.5%ficile N-terminal sequences and the sequences ofglutaraldehyde for 30 min. For extraction of S-

other previously characterized S-layer proteins.layer proteins, the cell envelopes were washed

It is known from studies on other bacterial patho-sequentially with 1 M NaCl, 2% Triton X-100

gens that the S-layer may contribute to the viru-(International Biotechnologies, Inc, CT, U.S.A.)

lence [7]. In fact, Aeromonas salmonicida becomesand three times with Tris–HCl pH 7.4 to remove

less virulent after loss of the S-layer [9].membranous contaminants.

In this study, the presence of two super-imposed regular arranged S-layers was dem-onstrated on all C. difficile strains examined

Electron microscopyincluding non-toxigenic strains isolated fromhealthy subjects. These results suggest that S-layer is a basic component of the bacterium but Freeze-etching

Whole bacterial cells were put on carriers andits possible role in the virulence is still unclear.However, since the S-layer covers the entire cell quickly frozen in Freon 22, which was partially

solidified by cooling with liquid nitrogen. Thesurface and is the immunodominant antigen ofthe cell, our data showing the large variability samples were cleaved at −100°C at a pressure

of 2–4×10−7 mbar in a Balzers BAF 300 freeze-of the S-layer proteins from different C. difficilestrains may highlight an important mechanism etch unit. The etching was carried out for 5 min

at−98°C. After shadowing with 2.5 nm of plat-to escape the host defence system. Further stud-ies will determine the functional characteristics inum carbon (at an angle of 45°C), and rep-

lication with 20 nm carbon deposits, cells wereof the C. difficile S-layer proteins.

370 M. Cerquetti et al.

digested by chlorox for 2 h and washed with centrifuged at 8000×g for 30 min and the pel-leted material was examined by electron micro-distilled water for 3 h. Samples were observed

with a Zeiss EM 902 transmission electron micro- scopy after negative staining.Purified S-layer proteins from C. difficile C253,scope at 80 KeV equipped with Electron Spec-

troscopy Imaging operating at De=0. Images obtained by reversed phase HPLC as previouslydescribed [21], were also tested for in vitro self-were directly captured in the Transmission Elec-

tron Microscope Zeiss EM902 by means of an assembly. The protein subunits (500 lg/ml) weretested either mixed with each other in a con-intensified camera model SIT66 and elaborated

by an image analyser model IBAS II (Kontron centration ratio of one-fold or each subunit aloneand sequentially dialysed and examined as de-Elektronik GMBH, Germany).scribed above.

Negative stainingCarbon film coated copper grids were floated

Extraction of S-layer proteins from wholeon drops of specimens for 1–5 min, blotted oncellsfilter paper and stained on drops of 2% phos-

photungstig acid. Grids were blotted and airBacterial cells were harvested by centrifugationdried.at 5800×g for 30 min and washed once with50 mM Tris–HCl, pH 7.4 at 4°C.Image processing

The washed pellet was treated with 8 M urea/To determine lattice constants and symmetriesTris–HCl, pH 8.3 added with 2 mM PMSF forof the crystalline surface arrays, flat areas of both1 h at 37°C and then centrifuged at 19 000×g fornegative stained and freeze-etched preparations30 min.were analysed by digital image analysis. Digital

Fast-Fourier-Transform (FFT) analysis was ap-plied to identify the 2-D symmetry and to meas- SDS-PAGEure the lattice spacing after an accuratecalibration performed with a standard sample Sodium dodecyl sulphate–polyacrylamide gel(catalase crystal). electrophoresis was performed following the

Statistical analysis, including at least 30 dif- method of Laemmli [25] on 12% polyacrylamideferent Freeze-etching observations for each gels in a Mini Protean II cell (BioRad Laborat-strain, was performed. In each symmetry pattern ories, CA, U.S.A.). After electrophoresis, gelsall first order points were identified and their were stained with Coomassie brilliant blue. Fordistance from the centre was measured. The molecular mass determination, the samples wereangles formed between two adjacent points and run in a slab gel (160×180×1.5 mm) in a verticalthe centre were also measured to distinguish cell (Bio-Rad Laboratories) and the low mo-between the lattice hexagonal model and the lecular weight standards (Bio-Rad Laboratories)lattice tetragonal (square/oblique) one. were used.

In vitro self-assembly of C. difficile C253 Detection of glycoproteinsS-layer proteins

Glycoproteins in the urea extract, separated onThe S-layer protein subunits of C. difficile C253, SDS-polyacrylamide gel, were directly detectedprototype strain we isolated from nosocomial on nitrocellulose blot by the DIG Glycan/proteinoutbreaks [3], were extracted from cell wall frag- double labelling kit (Boehringer Mannheimments by treatment with 8 M urea (International GmbH, Germany) according to the manu-Biotechnologies, Inc, CT, U.S.A.)/Tris–HCl facturer’s instruction. Fetuin and creatinase werepH 8.3 added with 2 mM PMNS (Sigma, St. used as positive and negative controls, re-Louis, MO, U.S.A.) for 1 h at 37°C and then spectively.ultracentrifuged at 100 000×g for 30 min at 4°C.

The urea extract, at a concentration of 400 lgprotein/ml, was dialysed overnight against sev- Antibody productioneral changes of the dialysis buffer (50 mM Tris–HCl, pH 7.4 with 2 mM PMSF added) in the Antisera to the S-layer proteins of C. difficile

C253 were obtained. Serum agaist the 47 kDapresence of 10 mM CaCl2. The dialysate was

Clostridium difficile surface layer proteins 371

2 Lyerly DM, Saum KE, MacDonald DK, Wilkins TD.protein was prepared in two adult New ZealandEffects of Clostridium difficile toxins given in-rabbits using antigen obtained by elution of thetragastrically to animals. Infect Immun 1985; 47: 349–52.

47 kDa band following the procedures pre-3 Cerquetti M, Pantosti A, Stefanelli P, Mastrantonio P.

viously reported for the antiserum to the 36 kDa Purification and characterization of an im-protein [3]. Each rabbit received two weekly munodominant 36 kDa antigen present on the cell sur-

face of Clostridium difficile. Microb Pathog 1992; 13: 271–9.doses for a total of 150 lg of protein. To increase4 Pantosti A, Cerquetti M, Viti F, Ortisi G, Mastrantoniothe specificity of the antiserum, antibodies to

P. Immunoblot analysis of serum immunoglobulin Gthe 36 kDa protein were removed by absorptionresponse to surface proteins of Clostridium difficile in

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